Modified Oligonucleotides and Applications Thereof

ABSTRACT

Disclosed, among other things, are primers containing certain modified nucleobases in the 3′ terminal region of the primers that provide reduced formation of primer-dimers during amplification reactions, and various methods of use thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/554,632, filed Sep. 4, 2009, to be issued as U.S. Pat. No. 7,807,376,which is a continuation of U.S. patent application Ser. No. 12/193,655,filed Aug. 18, 2008, now U.S. Pat. No. 7,585,649, issued on Sep. 8, 2009which is a continuation of U.S. patent application Ser. No. 11/372,984,filed Mar. 9, 2006, now U.S. Pat. No. 7,414,118, issued Aug. 19, 2008which is a continuation of U.S. patent application Ser. No. 11/106,045,filed Apr. 14, 2005, now abandoned, and claims a priority benefit under35 U.S.C. §119(e) from U.S. Patent Application No. 60/562,621, filedApr. 14, 2004, which are incorporated herein by reference.

The present teachings relate to nucleic acid amplification, for example,compounds and methods for application in the polymerase chain reaction(PCR).

Detecting the presence of target nucleic acids plays an important rolein a variety for applications in diverse fields, including: medicaldiagnostics, forensic science and genetic analysis. PCR is an example ofa nucleic acid amplification method that can provide a highly sensitivemeans for detecting the presence of target nucleic acids by selectiveamplification of a target nucleic acid sequence.

A significant problem with nucleic acid amplifications such as PCR isthe generation of non-specific amplification products. One example of anon-specific amplification process that can be problematic in PCRreactions is “primer-dimer” amplification. Primer-dimer amplificationcan result when, for example, the 3′ terminal region of a primer hassome degree of complimentarity with itself or another primer. Suchprimers will hybridize to one another to form primer-dimers.Amplification of the primer-dimer will then lead to primer-dimeramplicons that can in turn act as templates for further amplification.One outcome of such a process being a depletion of primers resulting inreduced sensitivity or even a failure to amplify the intended targetnucleic acid.

To complicate the problem, it is well known that the addition of a largeexcess of primers during PCR reactions allows even weak complimentarityat the 3′ terminal region to result in primer-dimer amplicons. As aresult there is a need to develop reagents and methods that suppressprimer-dimer formation in amplification reactions such as PCR.

It has now been found that, surprisingly, incorporation of certainmodified nucleobases in the 3′ terminal region of primers can havebeneficial impact on the formation of primer-dimer amplicons duringamplification reactions.

In some embodiments the present teachings provide for polynucleotidescomprising at least one modified pyrimidine nucleobase comprising thestructure

where X can be N or C, R¹ can be selected from —H, —F, —Cl, —Br, C₁-C₆alkyl, C₁-C₆ substituted alkyl, C₃-C₁₀ aryl, C₃-C₁₀ substituted aryl,—CF₃, —CF₂H, —CF₂CH₃, —CF₂CF₃, —CCl₃, —CN, —CHO, —CO₂R, —SO₃R, —PO₃RR,—C(O)NRR, azido, and —NO₂, and R² can be selected from —H, —F, —Cl, —Br,C₁-C₆ alkyl, C₁-C₆ substituted alkyl, C₃-C₁₀ aryl, C₃-C₁₀ substitutedaryl, —CF₃, —CF₂H, —CF₂CH₃, —CF₂CF₃, —CCl₃, —CN, —CHO, —CO₂R, —SO₃R,—PO₃RR, —C(O)NRR, azido, and —NO₂ where each R is independently —H,C₁-C₆ alkyl or C₃-C₁₀ aryl or alkylaryl, such that at least one of R¹ orR² is an electron withdrawing substituent or X is N, such that when X isN, R² is absent, and at least one said modified pyrimidine nucleobase isno more than 4 nucleotides from the 3′ terminus of the polynucleotide.In some embodiments, X can be C. In some embodiments, X is C and R² is—H. In some embodiments, X is C, R² is —H and R¹ can be selected from—F, —Cl, —Br, —CO₂R, —NO₂, —CHO, —COCH₃, —CF₃, —CF₂H, azido, or —CNwherein R is defined as above. In some embodiments, X is C, R² is —H andR¹ is —F. In some embodiments, X is C, R² is —H and R¹ is —CF₃. In someembodiments, X is C, R² is —H and R¹ is —CN. In some embodiments, X isC, R² is —H and R¹ is —Cl. In some embodiments, X is C, R² is —H and R¹is —CO₂R, wherein R is defined as above. In some embodiments, X is C, R²is —H and R¹ is —NO₂. In some embodiments, X is C, R² is —H and R¹ is—CHO. In some embodiments, X is C, R² is —H and R¹ is —COCH₃. In someembodiments, X is C, R² is —H and R¹ is —CF₂H. In some embodiments, X isC, R² is —H and R¹ is —Br.

In some embodiments, R¹ is —H. In some embodiments, R¹ is —H and X is C.In some embodiments, R¹ is —H, X is C and R² can be selected from —F,—Cl, —Br, —CO₂R, —NO₂, —CHO, —SO₃R, —PO₃RR—C(O)CH₃, —CF₃, —CF₂H, azido,and —CN, wherein R is defined as above. In some embodiments, R¹ is —H, Xis C and R² is —F. In some embodiments, R¹ is —H, X is C and R² is —Cl.In some embodiments, R¹ is —H, X is C and R² is —Br. In someembodiments, R¹ is —H, X is C and R² is —CO₂R, wherein R is defined asabove. In some embodiments, R¹ is —H, X is C and R² is —NO₂. In someembodiments, R¹ is —H, X is C and R² is —CHO. In some embodiments, R¹ is—H, X is C and R² is —SO₃R, wherein R is defined as above. In someembodiments, R¹ is —H, X is C and R² is —PO₃RR, wherein R is defined asabove. In some embodiments, R¹ is —H, X is C and R² is —C(O)CH₃. In someembodiments, R¹ is —H, X is C and R² is —CF₃. In some embodiments, R¹ is—H, X is C and R² is —CF₂H. In some embodiments, R¹ is —H, X is C and R²is —CN.

In some embodiments, X can be N. In some embodiments, X is N and R¹ isselected from —F, —Cl, —Br, —CHO, azido, and —CF₃. In some embodiments,X is N and R¹ is —F. In some embodiments, X is N and R¹ is —Cl. In someembodiments, X is N and R¹ is —Br. In some embodiments, X is N and R¹ is—CHO. In some embodiments, X is N and R¹ is —CF₃.

In some embodiments, the modified pyrimidine nucleobase can be selectedfrom

In some embodiments, the at least one modified pyrimidine nucleobase isno more than 3 nucleotides from the 3′ terminus of the polynucleotide.In some embodiments, the at least one modified pyrimidine nucleobase isno more than 2 nucleotides from the 3′ terminus of the polynucleotide.In some embodiments, the at least one modified nucleotide is the 3′terminal nucleotide of the polynucleotide.

In some embodiments, polynucleotides of the present teachings can beprimers in, for example, primer extension reactions. In someembodiments, polynucleotides of the present teachings are extendable atthe 3′-terminus.

In some embodiments, polynucleotides of the present teachings cancomprise at least one of a detectable label, a quencher or a minorgroove binder. In some embodiments, polynucleotides of the presentteachings can serve as probes.

In some embodiments, the present teachings provide for methods of primerextension comprising, annealing a polynucleotide primer to a denaturedDNA template such that, the polynucleotide primer anneals to acomplementary polynucleotide sequence on a strand of the denatured DNAtemplate to form a primer-template complex, and extending the primerportion of the primer-template complex to form a double strandedamplicon, wherein the polynucleotide primer is a primer according to thepresent teachings.

In some embodiments, the present teachings provide for methods of primerextension comprising, after the step of extending, denaturing the doublestranded amplicon. In some embodiments, the steps of annealing,extending and denaturing can be repeated at least one time. In someembodiments, the steps of annealing, extending and denaturing can berepeated at least 10 times. In some embodiments, the steps of annealing,extending and denaturing can be repeated at least 20 times. In someembodiments, the steps of annealing, extending and denaturing can berepeated at least 30 times. In some embodiments, the steps of annealing,extending and denaturing can be repeated at least 40 times.

In some embodiments, the present teachings provide for methods of primerextension, wherein the extending takes place in the presence ofextendable nucleotide triphosphates and non-extendable nucleotidetriphosphates to form DNA amplicon fragments. In some embodiments themethod of primer extension comprises, detecting the DNA ampliconfragments.

In some embodiments, the present teachings provide methods of primerextension comprising: i) annealing a first polynucleotide primer and asecond polynucleotide primer to a first and second strand of a denaturedDNA template such that, the first polynucleotide primer anneals to acomplementary oligonucleotide sequence on the first strand of thedenatured DNA template and the second polynucleotide primer anneals to acomplementary oligonucleotide sequence on the second strand of thedenatured DNA template to form a first and a second primer-templatecomplex, and ii) extending the primer portion of at least one of thefirst and second primer-template complex to form double stranded DNAamplicon, where at least one of the first polynucleotide primer or thesecond polynucleotide primer can be a polynucleotide according to thepresent teachings.

In some embodiments, the present teachings provide methods of primerextension comprising, prior to the step of annealing, forming a mixturecomprising a first polynucleotide primer, a second polynucleotideprimer, a DNA template, and other primer extension reagents. In someembodiments, the present teachings provide methods of primer extensioncomprising, after the step of forming but prior to the step ofannealing, denaturing the DNA template to form a first strand ofdenatured DNA template and a second denatured DNA template. In someembodiments, the present teachings provide methods of primer extensioncomprising, after the step of extending, denaturing the double strandedDNA amplicon.

In some embodiments, the steps of annealing, extending and denaturingthe double stranded DNA amplicon can optionally be repeated from 1-100times. Optionally, the steps of annealing, extending and denaturing thedouble stranded DNA amplicon can be repeated from 1-50 times. It will beunderstood that the present teachings encompass all possible ranges forrepeating the steps of annealing, extending and denaturing the doublestranded DNA amplicon between 1 and 100 times. That is, the steps ofannealing, extending and denaturing the double stranded DNA amplicon canbe repeated from 1 time up to 100 times and any number of times inbetween. For example, the range, of 1-10 will be understood to includeall possible ranges using all integers between 1 and 10, i.e.—1, 2, 3,4, 5, 6, 7, 8, 9, 10. In some embodiments, the steps of annealing,extending and denaturing the double stranded DNA amplicon can optionallybe repeated greater than 1 time. In some embodiments, the steps ofannealing, extending and denaturing the double stranded DNA amplicon canoptionally be repeated greater than 10 times. In some embodiments, thesteps of annealing, extending and denaturing the double stranded DNAamplicon can optionally be repeated greater than 20 times. In someembodiments, the steps of annealing, extending and denaturing the doublestranded DNA amplicon can optionally be repeated greater than 30 times.In some embodiments, the steps of annealing, extending and denaturingthe double stranded DNA amplicon can optionally be repeated greater than40 times. In some embodiments, the steps of annealing, extending anddenaturing the double stranded DNA amplicon can optionally be repeatedgreater than 50 times.

In some embodiments, the present teachings provide for methods of primerextension comprising, prior to the step of extending the primer portion,annealing a polynucleotide probe to a first or second strand of adenatured DNA template such that, the polynucleotide probe anneals to acomplementary polynucleotide sequence on the first strand of thedenatured DNA template and/or the polynucleotide probe anneals to acomplementary oligonucleotide sequence on the second strand of thedenatured DNA template. In some embodiments, the polynucleotide probecomprises at least one detectable label. In some embodiments, thepolynucleotide probe further comprises at least one of a quencher, aminor groove binder or both. In some embodiments, the polynucleotideprobe can be a polynucleotide of the present teachings.

In some embodiments, the present teachings provide methods ofoligonucleotide ligation comprising, i) forming a complex comprising afirst and a second polynucleotide strand annealed to a DNA template suchthat, the first polynucleotide strand anneals to a first complementarypolynucleotide sequence on the strand of the denatured DNA template andthe second polynucleotide strand anneals to a second complementarypolynucleotide sequence on the strand of the denatured DNA template,wherein the second complementary polynucleotide sequence on the strandof the denatured DNA template is located 5′ to the first complementarypolynucleotide sequence on the strand of the denatured DNA template, andii) forming a stable covalent bond between the first and secondpolynucleotide strands, wherein at least one of the first polynucleotidestrand or the second polynucleotide strand is a polynucleotide of thepresent teachings.

In some embodiments, the present teachings provide for methods fordetecting a target polynucleotide sequence comprising, (a) reacting atarget polynucleotide strand with a first probe pair comprising (i) afirst polynucleotide probe containing a sequence that is complementaryto a first target region in the target strand and (ii) a secondpolynucleotide probe comprising a sequence that is complementary to asecond target region in the target strand, wherein the second region islocated 5′ to the first region and overlaps the first region by at leastone nucleotide base, under conditions effective for the first and secondprobes to hybridize to the first and second regions in the targetstrand, respectively, forming a first hybridization complex, (b)cleaving the second probe in the first hybridization complex, to form asecond hybridization complex comprising the target strand, the firstprobe, and a first fragment of the second probe having a 5′ terminalnucleotide located immediately contiguous to a 3′ terminal nucleotide ofthe first probe, (c) ligating the first probe to the hybridized fragmentof the second probe to form a first ligated strand hybridized to thetarget strand, (d) denaturing the first ligated strand from the targetstrand, and (e) performing one or more additional cycles of steps (a)through (d), with the proviso that in the last cycle, step (d) isoptionally omitted, wherein at least one of the first probe, the secondprobe or both is a polynucleotide comprising at least one modifiedpyrimidine nucleobase comprising the structure

where X can be N or C, R¹ can be selected from —H, —F, —Cl, —Br, C₁-C₆alkyl, C₁-C₆ substituted alkyl, C₃-C₁₀ aryl, C₃-C₁₀ substituted aryl,—CF₃, —CF₂H, —CF₂CH₃, —CF₂CF₃, —CCl₃, —CN, —CHO, —CO₂R, —SO₃R, —PO₃RR,—C(O)NRR, azido, and —NO₂, and R² can be selected from —H, —F, —Cl, —Br,C₁-C₆ alkyl, C₁-C₆ substituted alkyl, C₃-C₁₀ aryl, C₃-C₁₀ substitutedaryl, —CF₃, —CF₂H, —CF₂CH₃, —CF₂CF₃, —CCl₃, —CN, —CHO, —CO₂R, —SO₃R,—PO₃RR, —C(O)NRR, azido, and —NO₂ where each R is independently —H,C₁-C₆ alkyl or C₃-C₁₀ aryl or alkylaryl, such that at least one of R¹ orR² is an electron withdrawing substituent or X is N, such that when X isN, R² is absent, and at least one said modified pyrimidine nucleobase isno more than 4 nucleotides from the 3′ terminus of the polynucleotide.In some embodiments, the modified pyrimidine nucleobase can be selectedfrom

In some embodiments, at least one said modified purine nucleobase is nomore than 2 nucleotides from the 3′ terminus of the first polynucleotideprobe, the second polynucleotide probe or both. In some embodiments, atleast one said modified purine nucleobase is no more than 1 nucleotidesfrom the 3′ terminus of the first polynucleotide probe, the secondpolynucleotide probe or both. In some embodiments, the modified purinenucleobase is the 3′ terminal nucleotide of the first polynucleotideprobe, the second polynucleotide probe or both.

In some embodiments, the first polynucleotide probe, the secondpolynucleotide probe or both comprise at least one of a detectablelabel, a quencher or a minor groove binder, or any combination thereof.In some embodiments, the first region overlaps the second region by onenucleotide base. In some embodiments, the 5′ end of the first probeterminates with a group other than a nucleotide 5′ phosphate group. Insome embodiments, the 5′ end of the first probe terminates with anucleotide 5′ hydroxyl group. In some embodiments, the 5′ end of thesecond probe terminates with a group other than a nucleotide 5′phosphate group. In some embodiments, the 5′ end of the second probeterminates with a nucleotide 5′ hydroxyl group. In some embodiments, the3′ end of the second probe terminates with a group other than anucleotide 3′ hydroxyl group. In some embodiments, the 3′ end of thesecond probe terminates with a nucleotide 3′ phosphate group.

In some embodiments, the present teachings provide methods for detectinga target polynucleotide sequence comprising, (a) reacting atarget-complementary strand with a second probe pair comprising (i) athird polynucleotide probe containing a sequence that is complementaryto a first region in the target-complementary strand and (ii) a fourthpolynucleotide probe containing a sequence that is complementary to asecond region in the target-complementary strand, wherein the secondregion is located 5′ to the first region and overlaps the first regionby at least one nucleotide base, under conditions effective for the forthe third and fourth probes to hybridize to the first and second regionsin the target-complementary strand, respectively, forming a thirdhybridization complex, (b) cleaving the fourth probe in the secondhybridization complex, to form a forth hybridization complex comprisingthe target-complementary strand, the third probe, and a first fragmentof the forth probe having a 5′ terminal nucleotide located immediatelycontiguous to a 3′ terminal nucleotide of the third probe, (c) ligatingthe third probe to the hybridized fragment of the fourth probe to form asecond ligated strand hybridized to the target-complementary strand, (d)denaturing the second ligated strand from the target-complementarystrand, and (e) performing one or more additional cycles of steps (a)through (d), with the proviso that in the last cycle, step (d) isoptionally omitted. In some embodiments, at least one of the third probeor the forth probe or both is a polynucleotide comprising at least onemodified pyrimidine nucleobase comprising the structure

where X can be N or C, R¹ can be selected from —H, —F, —Cl, —Br, C₁-C₆alkyl, C₁-C₆ substituted alkyl, C₃-C₁₀ aryl, C₃-C₁₀ substituted aryl,—CF₃, —CF₂H, —CF₂CH₃, —CF₂CF₃, —CCl₃, —CN, —CHO, —CO₂R, —SO₃R, —PO₃RR,—C(O)NRR, azido, and —NO₂, and R² can be selected from —H, —F, —Cl, —Br,C₁-C₆ alkyl, C₁-C₆ substituted alkyl, C₃-C₁₀ aryl, C₃-C₁₀ substitutedaryl, —CF₃, —CF₂H, —CF₂CH₃, —CF₂CF₃, —CCl₃, —CN, —CHO, —CO₂R, —SO₃R,—PO₃RR, —C(O)NRR, azido, and —NO₂ where each R is independently —H,C₁-C₆ alkyl or C₃-C₁₀ aryl or alkylaryl, such that at least one of R¹ orR² is an electron withdrawing substituent or X is N, such that when X isN, R² is absent, and at least one said modified pyrimidine nucleobase isno more than 4 nucleotides from the 3′ terminus of the polynucleotide.In some embodiments, the modified pyrimidine nucleobase can be selectedfrom

In some embodiments, at least one said modified purine nucleobase is nomore than 2 nucleotides from the 3′ terminus of the first polynucleotideprobe, the second polynucleotide probe or both. In some embodiments, atleast one said modified purine nucleobase is no more than 1 nucleotidefrom the 3′ terminus of the first polynucleotide probe, the secondpolynucleotide probe or both. In some embodiments, said modified purinenucleobase can be the 3′ terminal nucleotide of the first polynucleotideprobe, the second polynucleotide probe or both. In some embodiments, thefirst polynucleotide probe, the second polynucleotide probe or both cancomprise at least one of a detectable label, a quencher or a minorgroove binder, or any combination thereof. In some embodiments, the 5′end of the third probe optionally terminates with a group other than anucleotide 5′ phosphate group. In some embodiments, the 5′ end of thethird probe optionally terminates with a nucleotide 5′ hydroxyl group.In some embodiments, the 5′ end of the fourth probe optionallyterminates with a group other than a nucleotide 5′ phosphate group. Insome embodiments, the 5′ end of the fourth probe optionally terminateswith a nucleotide 5′ hydroxyl group. In some embodiments, the 5′ ends ofthe first, second, third and fourth probes optionally terminate with agroup other than a nucleotide 5′ phosphate group. In some embodiments,the 3′ end of the fourth probe optionally terminates with a group otherthan a nucleotide 3′ hydroxyl group. In some embodiments, the 3′ end ofthe fourth probe optionally terminates with a nucleotide 3′ phosphategroup. In some embodiments, at least one of the probes contains adetectable label. In some embodiments, the label can be a fluorescentlabel. In some embodiments, the label can be a radiolabel. In someembodiments, the label can be a chemiluminescent label. In someembodiments, the label can be an enzyme. In some embodiments, at leastone of the first probe and the third probe contains a detectable label.In some embodiments, each of the first probe and third probe contains adetectable label. In some embodiments, the detectable labels on thefirst probe and third probe are the same. In some embodiments, at leastone of the second probe and the fourth probe contains a detectablelabel. In some embodiments, each of the second probe and the fourthprobe contains a detectable label. In some embodiments, the second probeand fourth probe contain the same detectable label. In some embodiments,said cleaving produces a second fragment from the second probe whichdoes not associate with the second hybridization complex, and the methodfurther includes detecting said second fragment from the second probe.In some embodiments, said cleaving produces a second fragment from theforth probe which does not associate with the forth hybridizationcomplex, and the method further includes detecting said second fragmentfrom the forth probe. In some embodiments, at least one of the secondprobe and the fourth probe contains both (i) a fluorescent dye and (ii)a quencher dye which is capable of quenching fluorescence emission fromthe fluorescent dye when the fluorescent dye is subjected tofluorescence excitation energy, and said cleaving severs a covalentlinkage between the fluorescent dye and the quencher dye in the secondprobe and/or fourth probe, thereby increasing an observable fluorescencesignal from the fluorescent dye. In some embodiments, the second probeand the fourth probe each contain (i) a fluorescent dye and (ii) aquencher dye.

In some embodiments, methods of the present teachings for detectingtarget polynucleotide sequences method further include detecting bothsecond fragments. In some embodiments, the second fragment comprises oneor more contiguous nucleotides substantially non-complementary to thetarget strand. In some embodiments, the one or more contiguousnucleotides comprise 1 to 20 nucleotides.

In some embodiments, methods of the present teachings for detectingtarget polynucleotide sequences further include immobilizing the secondfragment on a solid support. In some embodiments, methods of the presentteachings for detecting target polynucleotide sequences further includesubjecting the second fragment to electrophoresis. In some embodiments,methods of the present teachings for detecting target polynucleotidesequences further include detecting the second fragment by massspectrometry. In some embodiments, methods of the present teachings fordetecting target polynucleotide sequences comprise detecting the secondfragment after the last cycle. In some embodiments, methods of thepresent teachings for detecting target polynucleotide sequences comprisedetecting the second fragment during or after a plurality of cycles. Insome embodiments, methods of the present teachings for detecting targetpolynucleotide sequences comprise detecting the second fragment duringall of the cycles.

In some embodiments, methods of the present teachings for detectingtarget polynucleotide sequences further include detecting the firsthybridization complex, the second hybridization complex, or both, afterat least one cycle. In some embodiments, the method further includesdetecting the third hybridization complex, the fourth hybridizationcomplex, or both, after at least one cycle. In some embodiments, themethod further includes detecting the first ligated strand, the secondligated strand, or both, after at least one cycle. In some embodiments,the detecting comprises an electrophoretic separation step.

In some embodiments, the present teachings provide for methods offragment analysis comprising: i) annealing an oligonucleotide primer toa denatured DNA template such that, the oligonucleotide primer annealsto a complementary oligonucleotide sequence on a strand of the denaturedDNA template to form a primer-template complex, ii) extending the primerportion of the primer-template complex in the presence ofdeoxyribonucleic acids and non-extendable ribonucleic acids to form DNAamplicon fragments, and iii) detecting the DNA amplicon fragments, wherethe oligonucleotide primer is an polynucleotide of the presentteachings.

Scheme 1 below illustrates an exemplary polynucleotide comprising aplurality, (x), of nucleotides, “N”, that may define a desirednucleotide sequence, wherein the subscripts 1, 2, 3 . . . x refer to theposition of the nucleotide in the primer relative to the 3′ end, and “ .. . ” indicates the possibility of one or more additional nucleotidesbetween N_(X) and N₇.

Scheme 1 5′-N_(x).......N₇N₆N₅N₄N₃N₂N₁-3′

Thus, N₁ is located at the 3′ terminus of the exemplary polynucleotide,and can be referred to as the 3′ terminal nucleotide of the exemplarypolynucleotide. Similarly, N₂ is located at the second nucleotideposition, and can be referred to as being 1 nucleotide from the 3′terminus. Similarly, N₃ is located at the third nucleotide position, andcan be referred to as being 2 nucleotides from the 3′ terminus.Similarly, N₄ is located at the fourth nucleotide position, and can bereferred to as being 3 nucleotides from the 3′ terminus. It is believedthat the effect of reduced primer-dimer formation that results fromincorporation of nucleobases of the present teachings into primers willdecrease in primers having no nucleotide of the present teachings anynearer the 3′-terminus than about the N₄ position.

As used herein, the terms oligonucleotide, polynucleotide and nucleicacid are used interchangeably to refer to single- or double-strandedpolymers of DNA, RNA or both including polymers containing modified ornon-naturally occurring nucleotides. In addition, the termsoligonucleotide, polynucleotide and nucleic acid refer to any other typeof polymer comprising a backbone and a plurality of nucleobases that canform a duplex with a complimentary polynucleotide strand bynucleobase-specific base-pairing. including, but not limited to, peptidenucleic acids (PNAs) which are disclosed in, for example, Nielsen etal., Science, 254:1497-1500 (1991), bicyclo DNA oligomers (Bolli et al.,Nucleic Acids Res., 24:4660-4667 (1996)) and related structures.

In some embodiments, polynucleotides of the present teachings cancomprise a backbone of naturally occurring sugar or glycosidic moieties,for example, 13-D-ribofuranose. In addition, in some embodiments,modified nucleotides of the present teachings can comprise a backbonethat includes one or more “sugar analogs”. As used herein, the term“sugar analog” refers to analogs of the sugar ribose. Exemplary ribosesugar analogs include, but are not limited to, substituted orunsubstituted furanoses having more or fewer than 5 ring atoms, e.g.,erythroses and hexoses and substituted or unsubstituted 3-6 carbonacyclic sugars. Typical substituted furanoses and acyclic sugars arethose in which one or more of the carbon atoms are substituted with oneor more of the same or different —R, —OR, —NRR or halogen groups, whereeach R is independently —H, (C₁-C₆) alkyl or (C₃-C₁₄) aryl. Examples ofunsubstituted and substituted furanoses having 5 ring atoms include butare not limited to 2′-deoxyribose, 2′-(C₁-C₆)-alkylribose,2′-(C₁-C₆)-alkoxyribose, 2′-(C₅-C₁₄)-aryloxyribose, 2′,3′-dideoxyribose,2′,3′-dideoxyribose, 2′-deoxy-3′-haloribose, 2′-deoxy-3′-fluororibose,2′-deoxy-3′-chlororibose, 2′-deoxy-3′-aminoribose,2′-deoxy-3′-(C₁-C₆)-alkylribose, 2′-deoxy-3′-(C₁-C₆)-alkoxyribose,2′-deoxy-3′-(C₅-C₁₄)-aryloxyribose,3′-(C₁-C₆)-alkylribose-5′-triphosphate,2′-deoxy-3′-(C₁-C₆)-alkylribose-5′-triphosphate,2′-deoxy-3′-(C₁-C₆)-alkoxyribose-5′-triphosphate,2′-deoxy-3′-(C₅-C₁₄)-aryl-oxyribose-5′-triphosphate,2′-deoxy-3′-haloribose-5′-triphosphate,2′-deoxy-3′-aminoribose-5′-triphosphate,2′,3′-dideoxyribose-5′-triphosphate or2′,3′-didehydroribose-5′-triphosphate. Further sugar analogs include butare not limited to, for example “locked nucleic acids” (LNAs), i.e.,those that contain, for example, a methylene bridge between C-4′ and anoxygen atom at C-2′, such as

that are described in Wengel, et al. WO 99/14226, incorporated herein byreference, and Wengel J., Acc. Chem. Res., 32:301-310 (1998).

In some embodiments, polynucleotides of the present teachings includethose in which the phosphate backbone comprises one or more “phosphateanalogs”. The term “phosphate analog” refers to analogs of phosphatewherein the phosphorous atom is in the +5 oxidation state and one ormore of the oxygen atoms are replaced with a non-oxygen moiety.Exemplary analogs include, but are not limited to, phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate,boronophosphates, and associated counterions, including but not limitedto H⁺, NH₄ ⁺, Na⁺, Mg⁺⁺ if such counterions are present. Polynucleotidesof the present teachings containing phosphate analogs can comprise, forexample, phosphorothioate linkages, methylphosphonates and/orphosphoroamidates (see, Chen et al., Nucl. Acids Res., 23:2662-2668(1995)). Combinations of polynucleotide linkages are also within thescope of the present teachings.

In some embodiments, polynucleotides described herein can beincorporated into PNA and DNA/PNA chimeras. Including, peptide nucleicacids (PNAs, also known as polyamide nucleic acids), see, for example,Nielsen et al., Science, 254:1497-1500 (1991). PNAs contain heterocyclicnucleobase units that are linked by a polyamide backbone instead of thesugar-phosphate backbone characteristic of DNA and RNA. PNAs are capableof hybridization to complementary DNA and RNA target sequences.Synthesis of PNA oligomers and reactive monomers used in the synthesisof PNA oligomers are described in, for example, U.S. Pat. Nos.5,539,082; 5,714,331; 5,773,571; 5,736,336 and 5,766,855. Alternateapproaches to PNA and DNA/PNA chimera synthesis and monomers for PNAsynthesis have been summarized in, for example, Uhlmann, et al., Angew.Chem. Int. Ed., 37:2796-2823 (1998).

In some embodiments, polynucleotides of the present teachings can rangein size from a few nucleotide monomers in length, e.g. from 5 to 80, tohundreds or thousands of nucleotide monomers in length. For example,polynucleotides of the present teachings can contain from 5 to 50nucleotides, 5 to 30 nucleotides, or 5 to 20 nucleotides. When, in someembodiments, polynucleotides of the present teachings contain, forexample, from 5 to 30 nucleotides, such a range includes all possibleranges of integers between 5 and 30, for example 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29an 30 nucleotides in length. Whenever a polynucleotide is represented bya sequence of letters, such as “ATGCCTG,” it will be understood that thenucleotides are in 5′ to 3′ order from left to right and that “A”denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxygaunosine, and T denotes thymidine, unless otherwise indicated.Additionally, whenever a polynucleotide of the present teachings isrepresented by a sequence of letters that includes an “X”, it will beunderstood that the “X” denotes a variable nucleotide monomer, where “X”is a nucleotide monomer other than “A”, “C”, “G” or “T”.

In some embodiments, polynucleotides of the present teachings can serveas primers in amplification reactions. As used herein, “primer” refersto a polynucleotide as defined herein having a 3′ terminus that isextendable by addition of one or more nucleotide monomers or by ligationof a ligation probe.

In some embodiments, polynucleotides of the present teachings cancomprise one or more nucleotides each independently comprising amodified nucleobase.

As used herein, the term “modified nucleobase” includes nucleobases thatdiffer from the naturally-occurring bases (e.g. A, G, C, T and U) byaddition and/or deletion of one or more functional groups, differencesin the heterocyclic ring structure (i.e., substitution of carbon for aheteroatom, or vice versa), and/or attachment of one or moresubstitutents capable of lowering the pKa of the N-3 imino proton of apyrimidine nucleobase such that the pK_(a) of the modified nucleobase islower than the pK_(a) of uridine. In some embodiments, the pK_(a) of animino proton on the modified nucleobase is <8. In some embodiments, thepK_(a) of an imino proton on the modified nucleobase is <7. Examples ofsubstituents capable of lowering the pK_(a) of and imino proton suitablefor use in connection with the present teachings include, but are notlimited to, any substituent that is capable of lowering the pK_(a) ofthe imino proton (e.g.—electron withdrawing) of the nucleobase such thatthe pK_(a) of the modified nucleobase is lower than the pK_(a) ofuridine. Examples of such substituents include but are not limited tosubstituted lower alkyl, substituted aryl —CN, —CF₃, —CHF₂, —CCl₃,—CO₂R, —CONR₂, —NO₂, halogen, fluorine, chlorine and bromine where eachR is independently —H, C₁-C₆ alkyl or C₃-C₁₀ aryl or alkylaryl.

In some embodiments, modified nucleobases for use with the presentteachings include modified pyrimidine nucleobases having the structure

wherein X can be N or C, R₁ is selected from halogen, fluorine,chlorine, bromine, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₃-C₁₀ aryl,substituted C₃-C₁₀ aryl, —CF₃, —CF₂H, —CF₂CH₃, —CF₂CF₃, —CCl₃, —CN,—CHO, —CO₂R, —SO₃R, —PO₃R₂, —C(O)NR₂, azido, and —NO₂, and R₂ isselected from halogen, fluorine, chlorine, bromine, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₃-C₁₀ aryl, substituted C₃-C₁₀ aryl, —CF₃,—CF₂H, —CF₂CH₃, —CF₂CF₃, —CCl₃, —CN, —CHO, —CO₂R, —SO₃R, —PO₃R₂,—C(O)NR₂, azido, and —NO₂ where each R is independently —H, C₁-C₆ alkylor C₃-C₁₀ aryl or alkylaryl, for example, benzyl.

As used herein, “alkyl” refers to a saturated or unsaturated, branched,straight-chain or cyclic monovalent hydrocarbon radical derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane, alkene or alkyne. Typical alkyl groups include, but are notlimited to, methyl (methanyl); ethyls such as ethanyl, ethenyl, ethynyl;propyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl,prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls suchas butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, but-1-en-1-yl,but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Furthermore, cyclicalkyl groups may optionally have one or more ring carbon atomssubstituted by a heteroatom selected from O, S or N. Examples of such“heteroalkyl” groups include, but are not limited to morpholine, pyranand the like.

As used herein, “aryl” refers to a monovalent aromatic hydrocarbonradical derived by the removal of one hydrogen atom from a single carbonatom of an aromatic ring system. Typical aryl groups include, but arenot limited to, radicals derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In someembodiments, the aryl group is (C₅-C₂₀) aryl or (C₅-C₁₀) aryl. Furtheraryl groups are phenyl (C₆ aryl) and naphthyl (C₁₀ aryl).

As used herein, “substituted” as in “substituted alkyl”, “substitutedaryl” or “substituted alkylaryl”, means that the alkyl, aryl, amine,cyclic alkyl or phenyl moiety is substituted by one or moresubstituents. Such substituents include, but are not limited to, —F,—Cl, —Br, —CF₃, —CCl₃, —CN, —CHO, —CO₂R, —SO₃R, —PO₃RR, —C(O)NRR and—NO₂ where each R is independently —H, C₁-C₆ alkyl, C₃-C₁₀ aryl oralkylaryl.

In some embodiments, modified nucleobases for use in the presentteachings include, but are not limited to, 5-cyanouracil (5-CN—U),5-fluorouracil (5-F—U) and 5-trifluormethyluracil (5-CF₃—U).

Examples of nucleosides/tides including an electron withdrawingsubstituent as described above include, but are not limited to,5-cyano-2′-deoxyuridine, 5-fluoro-2′-deoxyuridine and5-trifluormethyl-2′-deoxyuridine, 5-cyanouridine, 5-fluorouridine and5-trifluoromethyluridine.

The pK_(a) value of the N-3 imino proton of 5-cyano-2′-deoxyuridine and5-fluoro-2′-deoxyuridine have been reported in the literature as beingabout 6.50 and about 7.7 respectively (Ishikawa, R. et al., MagneticResonance in Chemistry, v. 39, S159-S165 (2001)). In contrast, thepK_(a) of the imino proton in uridine is ˜9.8. 5-cyano-2′-deoxyuridinecan be prepared according to published literature procedures (see, forexample, Hampton, et al., J. Med. Chem., v.22 (6), 621-631 (1979)).

It will be understood that substituted for use in connection with thepresent teachings are well known in the art. Further examples ofmodified nucleobases and nucleosides/tides and the preparation thereof,having various X, R¹ and R² substitution patterns for use in connectionwith the present teachings include, but are not limited to, thosedescribed in, Felczak, K., et al. J. Med. Chem., v.39, 1720-1728 (1996),Mertes, M., et al., J. Med. Chem., v.9, 876-81 (1966), Mertes, M., etal., J. Med. Chem., 619 (1963), Khomov-Borisov, N., et al., ZhurnalObshchei Khimii, v.27, 2518-21 (1957), Giner-Sorolla, A., J. Am. Chem.Soc., v.80, 5744-52 (1958), Wempen, I., et al., J. Med. Chem., 207-9(1964), Greenbaum, S., et al. J. Am. Chem. Soc., v.76, 2899-2902 (1954),Vissen, B., et al., J. Biol. Chem., v.171, 377-81 (1947) and Asburn, W.,J. Org. Chem., 31(7), 2215-19 (1966), Honjo, M., et al., Chem. & Pharm.Bull., 35(8), 3227-34 (1987), Ueda, T., et al., Chem. & Pharm. Bull.,23(11), 2614-19 (1975), Tanaka, H., et al., Tetrahedron, 38(17), 2635-42(1982), Groziak, M. P., et al., J. Org. Chem., 58(15), 4054-60 (1993),Tanaka, H., et al., Tetrahedron, 41(5), 861-6 (1985), De Zeeuw, J. R.,et al., J. of Antibiotics, 22(8), 386-7 (1969), Beckvermit, J. T., etal., U.S. Pat. No. 6,020,483, Matsuda, A., et al., Bioorg. & Med. Chem.Letters, 3(12), 2751-4 (1993), Giziewicz, J., et al., J. Org. Chem.,64(6), 2149-51 (1999), Guerniou, V. et al., Nucleosides, Nucleotides &Nucleic Acids, 22(5-8), 1073-75 (2003), Barr, P. J., et al. Tetrahedron,36(9), 1269-73, Kampf, A., et al., J. Med. Chem., 19(7), 909-15 (1976),Matulic-Adamic, J., et al., J. Chem. Soc., Chem. Comm., v.21, 1535-6(1985), Haginoya, N., et al., Bioconjugate Chem., 8(3), 271-80 (1997),Kalman, T., et al., Nucleic Acid Chem, v.4, 84-6 (1991), Cody, V., etal. Nucleosides & Nucleotides, 4(5), 587-94 (1985), and references citedtherein.

Further examples of commercially available and non-commerciallyavailable nucleobases and nucleosides/tides for use in connection withthe present teachings and methods of making them can be found in avariety of databases that are known in the art, including Scifinder®Scholar, Chemical Abstracts®, and the like. It will be understood thatfurther nucleosides and nucleotides for use in connection with thepresent teachings will be readily accessible to those of skill in theart by chemical synthetic methods well known in the art in combinationwith or independent of the teachings provided herein and in thereferences cited above.

All tautomeric forms of naturally occurring bases, modified bases andbase analogues may be included in oligonucleotides of the presentteachings.

Accordingly, polynucleotides having any combination of normal bases,modified pyrimidine nucleobases, universal bases, sugar modifications,or backbone modifications of DNA, PNA or DNA/PNA chimeras are within thescope of the present teachings.

In some embodiments polynucleotides of the present teachings can beconjugated to at least one detectable label, nonfluorescent quencherand/or at least one stabilizing moiety. In some embodimentspolynucleotides of the present teachings that are conjugated to at leastone detectable label, nonfluorescent quencher and/or at least onestabilizing moiety can serve as probes or primers in amplificationreactions.

The term “detectable label” refers to any moiety that, when attached topolynucleotides of the present teachings, render such polynucleotidesdetectable using known detection means. Exemplary detectable labelsinclude, but are not limited to, fluorophores, chromophores,radioisotopes, spin-labels, enzyme labels or chemiluminescent labelsthat allow for direct detection of a labeled compound by a suitabledetector, or a binding pair, for example, a ligand, such as an antigenor biotin, that can bind specifically with high affinity to a detectableanti-ligand, such as a labeled antibody or avidin. In some embodimentsthe labels can be fluorescent dyes, such as fluorescein or rhodaminedyes or fluorescent dye pairs, such as FRET dyes.

In some embodiments, polynucleotides of the present teachings cancomprise one or more “nonfluorescent quencher” moieties. As used herein,“nonfluorescent quencher” includes but is not limited to, for example,particular azo dyes (such as DABCYL or DABSYL dyes and their structuralanalogs), triarylmethane dyes such as malachite green or phenol red,4′,5′-diether substituted fluoresceins (U.S. Pat. No. 4,318,846),asymmetric cyanine dye quenchers (see, Lee et al., U.S. Pat. No.6,080,868 and Lee, et al., U.S. Pat. No. 6,348,596), or nonfluorescentderivatives of 3- and/or 6-amino xanthene that is substituted at one ormore amino nitrogen atoms by an aromatic or heteroaromatic ring system(Haugland, et al., U.S. Pat. No. 6,399,392).

“Nonfluorescent”, as used herein, indicates that the fluorescenceefficiency of the quenching moiety in an assay solution as described forany of the methods herein is less than or equal to 5 percent emission atemission-λ_(max). In some embodiments, less than or equal to 1 percentemission at emission-λ_(max). In some embodiments of the presentteachings, the covalently bound quenching moiety exhibits a quantumyield of less than about 0.1 percent emission at emission-λ_(max). Insome embodiments, less than about 0.01 percent emission atemission-λ_(max).

In some embodiments, polynucleotides of the present teachings can beconjugated to at least one “stabilizing moiety”. As used herein, theterm “stabilizing moiety” refers to moieties that include but are notlimited to minor groove binder (MGB) moieties. A variety of suitableminor groove binders have been described in the literature. See, forexample, Kutyavin, et al. U.S. Pat. No. 5,801,155; Wemmer, D. E., andDervan P. B., Current Opinion in Structural Biology, 7:355-361 (1997);Walker, W. L., Kopka, J. L. and Goodsell, D. S., Biopolymers, 44:323-334(1997); Zimmer, C & Wahnert, U. Prog. Biophys. Molec. Bio. 47:31-112(1986) and Reddy, B. S. P., Dondhi, S. M., and Lown, J. W., Pharmacol.Therap., 84:1-111 (1999).

Suitable methods for attaching MGBs (as well as reporter groups such asfluorophores and quenchers described above) through linkers topolynucleotides are described in, for example, U.S. Pat. Nos. 5,512,677;5,419,966; 5,696,251; 5,585,481; 5,942,610 and 5,736,626. Minor groovebinders include, for example, the trimer of3-carbamoyl-1,2-dihydro-(3-H7)-pyrrolo[3,2-e]indole-7-carboxylate(CDPI₃) and the pentamer of N-methylpyrrole-4-carbox-2-amide (MPC₅).Additional MGB moieties are disclosed in U.S. Pat. No. 5,801,155. Incertain embodiments, the MGBs can have attached watersolubility-enhancing groups (e.g., sugars or amino acids).

Polynucleotides of the present teachings can find use as primers and/orprobes in, for example, polynucleotide chain extension or ligationreactions. As used herein, “chain extension reaction” refers to primerextension reactions in which at least one polynucleotide of the presentteachings (e.g.—as primers or ligation probes) can be annealed to atleast one DNA template strand. After the step of annealing in apolynucleotide chain extension reaction, the primer can then be extendedby at least one nucleotide to form an amplicon or extension product.Alternatively, after the step of annealing in a polynucleotide chainextension reaction, the primer can be ligated to a second ligation probeto form a ligation product. The present teachings encompass all possiblechain extension reactions including, but not limited to, polymerasechain reaction (PCR), nested PCR, asynchronous PCR, real time PCR,TaqMan assays, DNA sequencing, cycled DNA sequencing, oligonucleotideligation assay (OLA), and fragment analysis, described in, for example,The PCR Technique: DNA Sequencing II, Eaton Publishing Co. (1997),Genome Analysis, A Laboratory Manual Volume 1: Analyzing DNA, Birren,B., Green, E., Klapholz, S., Myers, R. M. and Roskams, J. Eds., ColdSpring Harbor Laboratory Press (1997), Innis, M. et al., PCR Protocols:A Guide to Methods and Applications, Academic Press (1989), Chen, C. etal., U.S. Patent Application Pub. No. 2003/0207266 A1, Erlich, et al.,U.S. Pat. No. 5,314,809, U.S. Pat. No. 6,221,606 and Bi, W., et al.,U.S. Pat. No. 6,511,810. Oligonucleotides of the present teachings canbe used in any of the above primer extension reactions as either primersor probes where each is appropriate.

In some embodiments, the present teachings provide for methods of primerextension comprising, annealing a polynucleotide primer to a denaturedDNA template such that, the polynucleotide primer anneals to acomplementary polynucleotide sequence on a strand of the denatured DNAtemplate to form a primer-template complex, and extending the primerportion of the primer-template complex to form a double strandedamplicon, wherein the polynucleotide primer is a primer according to thepresent teachings.

In some embodiments, the present teachings provide for methods of primerextension comprising, after the step of extending, denaturing the doublestranded amplicon. In some embodiments, the steps of annealing,extending and denaturing can be repeated at least one time. In someembodiments, the steps of annealing, extending and denaturing can berepeated at least 10 times. In some embodiments, the steps of annealing,extending and denaturing can be repeated at least 20 times. In someembodiments, the steps of annealing, extending and denaturing can berepeated at least 30 times. In some embodiments, the steps of annealing,extending and denaturing can be repeated at least 40 times.

In some embodiments, the present teachings provide for a method ofprimer extension comprising i) annealing a first polynucleotide primerand a second polynucleotide primer to a first and second strand of adenatured DNA template such that, the first polynucleotide primeranneals to a complementary oligonucleotide sequence on the first strandof the denatured DNA template and the second polynucleotide primeranneals to a complementary oligonucleotide sequence on the second strandof the denatured DNA template to form a first and a secondprimer-template complex, and ii) extending the primer portion of atleast one of the first and second primer-template complex to form doublestranded DNA amplicon, wherein at least one of the first polynucleotideprimer or the second polynucleotide primer can be a polynucleotide ofthe present teachings. In some embodiments, prior to the step ofannealing, the method can include the step of forming a mixturecomprising a first polynucleotide primer, a second polynucleotideprimer, a DNA template, and other primer extension reagents, including,for example buffers and polymerases. In some embodiments, polymerasesfor use in the present teachings can comprise at least one thermostablepolymerase, including, but not limited to, Taq, Pfu, Vent, Deep Vent,Pwo, UITma, and Tth polymerase and enzymatically active mutants andvariants thereof. Such polymerases are well known and/or arecommercially available. Descriptions of polymerases can be found, amongother places, at the world wide web URL:the-scientist.library.upenn.edu/yr1998/jan/profil e 1_(—)980105.html.

In some embodiments, after the step of forming but prior to the step ofannealing, the method can include the step of denaturing the DNAtemplate to form a first strand of denatured DNA template and a seconddenatured DNA template. In some embodiments, after the step ofextending, denaturing the double stranded DNA amplicon. In someembodiments, the steps of annealing, extending and denaturing the doublestranded DNA amplicon can be repeated from 1-100 times. In someembodiments, the steps of annealing, extending and denaturing the doublestranded DNA amplicon can be repeated from 10-100 times. In someembodiments, the steps of annealing, extending and denaturing the doublestranded DNA amplicon can be repeated from 20-100 times. In someembodiments, the steps of annealing, extending and denaturing the doublestranded DNA amplicon can be repeated from 30-100 times. In someembodiments, the steps of annealing, extending and denaturing can berepeated at least one time. In some embodiments, the steps of annealing,extending and denaturing can be repeated at least 10 times. In someembodiments, the steps of annealing, extending and denaturing can berepeated at least 20 times. In some embodiments, the steps of annealing,extending and denaturing can be repeated at least 30 times. In someembodiments, the steps of annealing, extending and denaturing can berepeated at least 40 times.

In some embodiments, the present teachings provide for methods of primerextension comprising, prior to the step of extending the primer portion,annealing a polynucleotide probe to a first or second strand of adenatured DNA template such that, the polynucleotide probe anneals to acomplementary polynucleotide sequence on the first strand of thedenatured DNA template and/or the polynucleotide probe anneals to acomplementary oligonucleotide sequence on the second strand of thedenatured DNA template. In some embodiments, the polynucleotide probecomprises at least one detectable label. In some embodiments, thepolynucleotide probe further comprises at least one of a quencher, aminor groove binder or both. In some embodiments, the polynucleotideprobe can be a polynucleotide of the present teachings.

In some embodiments, the present teachings provide “fragment analysis”or “genetic analysis” methods, wherein labeled polynucleotide fragmentscan be generated through template-directed enzymatic synthesis usinglabeled primers or nucleotides, the fragments can be subjected to asize-dependent separation process, e.g., electrophoresis orchromatography; and, the separated fragments can be detected subsequentto the separation, e.g., by laser-induced fluorescence. In someembodiments, multiple classes of polynucleotides are separatedsimultaneously and the different classes are distinguished by spectrallyresolvable labels.

In some embodiments, the present teachings provide a method of fragmentanalysis in which fragment classes can be identified and defined interms of terminal nucleotides so that a correspondence is establishedbetween the four possible terminal bases and the members of a set ofspectrally resolvable dyes. Such sets are readily assembled from thefluorescent dyes known in the art by measuring emission and absorptionbandwidths using commercially available spectrophotometers. In someembodiments, fragment classes are formed through chain terminationmethods of DNA sequencing, i.e., dideoxy DNA sequencing, or Sanger-typesequencing.

Sanger-type sequencing involves the synthesis of a DNA strand by a DNApolymerase in vitro using a single-stranded or double-stranded DNAtemplate whose sequence is to be determined. Synthesis is initiated at adefined site based on where an oligonucleotide primer anneals to thetemplate. The synthesis reaction is terminated by incorporation of anucleotide analog that will not support continued DNA elongation.Exemplary chain-terminating nucleotide analogs include the2′,3′-dideoxynucleoside 5′-triphosphates (ddNTPs) which lack the 3′-OHgroup necessary for 3′ to 5′ DNA chain elongation. When properproportions of dNTPs (2′-deoxynucleoside 5′-triphosphates) and one ofthe four ddNTPs are used, enzyme-catalyzed polymerization will beterminated in a fraction of the population of chains at each site wherethe ddNTP is incorporated. If labeled primers or labeled ddNTPs are usedfor each reaction, the sequence information can be detected byfluorescence after separation by high-resolution electrophoresis. In thechain termination method, dyes of the invention can be attached toeither sequencing primers or dideoxynucleotides. In the method,fluorescent dye molecules can be linked to a complementary functionalityat, for example, the 5′-terminus of a primer, e.g. following theteaching in Fung, et al., U.S. Pat. No. 4,757,141; on the nucleobase ofa primer; or on the nucleobase of a dideoxynucleotide, e.g. via thealkynylamino linking groups disclosed by Hobbs et al, in European PatentApplication No. 87305844.0, and Hobbs et al., J. Org. Chem., 54: 3420(1989) incorporated herein by reference.

In some embodiments, labeled polynucleotides can be preferably separatedby electrophoretic procedures as disclosed in, for example, Rickwood andHames, Eds., Gel Electrophoresis of Nucleic Acids: A Practical Approach,IRL Press Limited, London, 1981; Osterman, Methods of Protein andNucleic Acid Research, Vol. 1 Springer-Verlag, Berlin, 1984; or U.S.Pat. Nos. 5,374,527, 5,624,800 and/or 5,552,028. In some embodiments,the type of electrophoretic matrix can be crosslinked or uncrosslinkedpolyacrylamide having a concentration (weight to volume) of betweenabout 2-20 weight percent. In some embodiments, the polyacrylamideconcentration is between about 4-8 percent. In some embodiments, forexample in DNA sequencing, the electrophoresis matrix can include adenaturing agent, e.g., urea, formamide, and the like. Detailedprocedures for constructing such matrices are given by, for example,Maniatis et al., “Fractionation of Low Molecular Weight DNA and RNA inPolyacrylamide Gels Containing 98% Formamide or 7 M Urea,” in Methods inEnzymology, 65: 299-305 (1980); Maniatis et al., “Chain LengthDetermination of Small Double- and Single-Stranded DNA Molecules byPolyacrylamide Gel Electrophoresis,” Biochemistry, 14: 3787-3794 (1975);Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, pgs. 179-185 (1982); and ABI PRISM™ 377 DNASequencer User's Manual, Rev. A, January 1995, Chapter 2 (AppliedBiosystems, Foster City, Calif.). It will be understood that optimalelectrophoresis conditions, for example, polymer concentration, pH,temperature, and concentration of denaturing agent, employed in aparticular separation depends on many factors, including the size rangeof the nucleic acids to be separated, their base compositions, whetherthey are single stranded or double stranded, and the nature of theclasses for which information is sought by electrophoresis.

Subsequent to electrophoretic separation, labeled polynucleotides can bedetected by, for example, measuring the fluorescence emission from a dyeon the labeled polynucleotides. To perform such detection, the labeledpolynucleotides are illuminated by standard means, such as highintensity mercury vapor lamps, lasers, or the like. In some embodiments,the illumination means is a laser having an illumination beam at awavelength above about 600 nm. In some embodiments, thedye-polynucleotides are illuminated by laser light generated by a He—Negas laser or a solid-state diode laser. After illumination, fluorescenceintensity of the labeled polynucleotide can be measured by alight-sensitive detector, such as a photomultiplier tube, chargedcoupled device, or the like. Exemplary electrophoresis detection systemsare described elsewhere, e.g., U.S. Pat. Nos. 5,543,026; 5,274,240;4,879,012; 5,091,652 and 4,811,218.

In some embodiments, the present teachings provide for a method offragment analysis comprising, annealing an polynucleotide primer to adenatured DNA template, such that the polynucleotide primer anneals to acomplementary polynucleotide sequence on a strand of the denatured DNAtemplate to form a primer-template complex, extending the primer portionof the primer-template complex in the presence of extendable nucleotidetriphosphates and non-extendable nucleotide triphosphates to form DNAamplicon fragments and detecting the DNA amplicon fragments. In someembodiments, the polynucleotide primer can be an oligonucleotide of thepresent teachings. Further embodiments of fragment analysis methods inaccordance with the present teachings can be found in, for example, U.S.Pat. No. 6,221,606 incorporated herein by reference.

As used herein, “ligation reaction” refers to reactions in which allelespecific ligation probes are annealed to at least one DNA templatestrand to form a probe-template complex. After the step of annealing, acovalent bond is then formed between the probe and the secondoligonucleotide fragment by a ligation agent to form a ligation product.A ligation agent according to the present invention may comprise anynumber of enzymatic or chemical (i.e., non-enzymatic) agents. Forexample, ligase is an enzymatic ligation agent that, under appropriateconditions, forms phosphodiester bonds between the 3′-OH and the5′-phosphate of adjacent polynucleotides. Temperature-sensitive ligases,include, but are not limited to, bacteriophage T4 ligase, bacteriophageT7 ligase, and E. coli ligase. Thermostable ligases include, but are notlimited to, Taq ligase, Tth ligase, and Pfu ligase. Thermostable ligasemay be obtained from thermophilic or hyperthermophilic organisms,including but not limited to, prokaryotic, eucaryotic, or archaelorganisms. Some RNA ligases may also be employed in the methods of theinvention.

Chemical ligation agents include, without limitation, activating,condensing, and reducing agents, such as carbodiimide, cyanogen bromide(BrCN), N-cyanoimidazole, imidazole,1-methylimidazole/carbodiimide/cystamine, dithiothreitol (DTT) andultraviolet light. Autoligation, i.e., spontaneous ligation in theabsence of a ligating agent, is also within the scope of the invention.Detailed protocols for chemical ligation methods and descriptions ofappropriate reactive groups can be found, among other places, in Xu etal., Nucleic Acid Res., 27:875-81 (1999); Gryaznov and Letsinger,Nucleic Acid Res., 21:1403-08 (1993); Gryaznov et al., Nucleic AcidRes., 22:2366-69 (1994); Kanaya and Yanagawa, Biochemistry, 25:7423-30(1986); Luebke and Dervan, Nucleic Acids Res., 20:3005-09 (1992);Sievers and von Kiedrowski, Nature, 369:221-24 (1994); Liu and Taylor,Nucleic Acids Res., 26:3300-04 (1999); Wang and Kool, Nucleic AcidsRes., 22:2326-33 (1994); Purmal et al., Nucleic Acids Res., 20:3713-19(1992); Ashley and Kushlan, Biochemistry, 30:2927-33 (1991); Chu andOrgel, Nucleic Acids Res., 16:3671-91 (1988); Sokolova et al., FEBSLetters, 232:153-55 (1988); Naylor and Gilham, Biochemistry, 5:2722-28(1966); U.S. Pat. No. 5,476,930; and Royer, EP 324616B1). In someembodiments, the ligation agent is an “activating” or reducing agent. Itwill be appreciated that if chemical ligation is used, the 3′ end of thefirst probe and the 5′ end of the second probe should includeappropriate reactive groups to facilitate the ligation.

In some embodiments, the present teachings provide methods ofoligonucleotide ligation comprising, i) forming a complex comprising afirst and a second polynucleotide strand annealed to a DNA template suchthat, the first polynucleotide strand anneals to a first complementarypolynucleotide sequence on the strand of the denatured DNA template andthe second polynucleotide strand anneals to a second complementarypolynucleotide sequence on the strand of the denatured DNA template,wherein the second complementary polynucleotide sequence on the strandof the denatured DNA template is located 5′ to the first complementarypolynucleotide sequence on the strand of the denatured DNA template, andii) forming a stable covalent bond between the first and secondpolynucleotide strands, wherein at least one of the first polynucleotidestrand or the second polynucleotide strand is a polynucleotide of thepresent teachings.

In some embodiments, the present teachings provide a method ofoligonucleotide ligation comprising, annealing a first oligonucleotideto a strand of a denatured DNA template such that, the firstoligonucleotide anneals to a complementary oligonucleotide sequence onthe strand of the denatured DNA template to form a firstoligonucleotide-template complex, annealing a second oligonucleotide toan oligonucleotide sequence on the first oligonucleotide-templatecomplex that is complementary to the second oligonucleotide to form asecond oligonucleotide-template complex, wherein the secondoligonucleotide anneals to the first oligonucleotide-template complex sothat the 3′-terminus of the first oligonucleotide and the 5′-terminus ofthe second oligonucleotide are associated with adjacent nucleotides ofthe denatured DNA template, and forming a stable covalent bond betweenthe 3′-terminus of the first oligonucleotide and the 5′-terminus of thesecond oligonucleotide. In some embodiments, at least one of the firstoligonucleotide or the second oligonucleotide can be an oligonucleotideof the present teachings.

In some embodiments, the present teachings provide for a method fordetecting a target polynucleotide sequence comprising (a) reacting atarget polynucleotide strand and a target-complementary strand with afirst probe pair and a second probe pair, the first probe paircomprising (i) a first polynucleotide probe containing a sequence thatis complementary to a first target region in the target strand and (ii)a second polynucleotide probe comprising a sequence that iscomplementary to a second target region in the target strand, whereinthe second region is located 5′ to the first region and overlaps thefirst region by at least one nucleotide base, and the second probe paircomprising (i) a third polynucleotide probe containing a sequence thatis complementary to a first region in the target-complementary strandand (ii) a fourth polynucleotide probe containing a sequence that iscomplementary to a second region in the target-complementary strand,wherein the second region is located 5′ to the first region and overlapsthe first region by at least one nucleotide base, under conditionseffective for the first and second probes to hybridize to the first andsecond regions in the target strand, respectively, forming a firsthybridization complex, and for the third and fourth probes to hybridizeto the first and second regions in the target-complementary strand,respectively, forming a second hybridization complex, (b) cleaving thesecond probe in the first hybridization complex, and the fourth probe inthe second hybridization complex, to form (i) a third hybridizationcomplex comprising the target strand, the first probe, and a firstfragment of the second probe having a 5′ terminal nucleotide locatedimmediately contiguous to a 3′ terminal nucleotide of the first probe,and (ii) a fourth hybridization complex comprising thetarget-complementary strand, the third probe, and a first fragment ofthe fourth probe having a 5′ terminal nucleotide located immediatelycontiguous to a 3′ terminal nucleotide of the third probe, (c) ligatingthe first probe to the hybridized fragment of the second probe to form afirst ligated strand hybridized to the target strand, and ligating thethird probe to the fragment of the fourth probe to form a second ligatedstrand hybridized to the target-complementary strand, (d) denaturing thefirst ligated strand from the target strand and the second ligatedstrand from the target-complementary strand, and (e) performing one ormore additional cycles of steps (a) through (d), with the proviso thatin the last cycle, step (d) is optionally omitted.

In some embodiments, the first region can overlap the second region byone nucleotide base. In some embodiments, the 5′ ends of the first andthird probes can terminate with a group other than a nucleotide 5′phosphate group. In some embodiments, the 5′ ends of the first and thirdprobes can terminate with a nucleotide 5′ hydroxyl group. In someembodiments, the 5′ ends of the second and fourth probes can terminatewith a group other than a nucleotide 5′ phosphate group. In someembodiments, the 5′ ends of the second and fourth probes can terminatewith a nucleotide 5′ hydroxyl group. In some embodiments, the 5′ ends ofthe first, second, third and fourth probes can each independentlyterminate with a group other than a nucleotide 5′ phosphate group. Insome embodiments, the 3′ ends of the second and fourth probes can eachindependently terminate with a group other than a nucleotide 3′ hydroxylgroup. In some embodiments, the 3′ ends of the second and fourth probescan terminate with a nucleotide 3′ phosphate group.

In some embodiments, at least one of the probes can contain a detectablelabel. In some embodiments, the label can be a fluorescent label. Insome embodiments, the label can be a radiolabel. In some embodiments,the label can be a chemiluminescent label. In some embodiments, thelabel can be an enzyme. In some embodiments, at least one of the firstprobe and the third probe can contain a detectable label. In someembodiments, each of the first probe and third probe can contain adetectable label. In some embodiments, the detectable labels on thefirst probe and third probe can be the same. In some embodiments, atleast one of the second probe and the fourth probe can contain adetectable label. In some embodiments, each of the second probe and thefourth probe contains a detectable label. In some embodiments, thesecond probe and fourth probe can contain the same detectable label.

In some embodiments, the step of cleaving produces a second fragmentfrom the second probe which does not associate with the thirdhybridization complex, and the method further includes detecting saidsecond fragment.

In some embodiments, at least one of the second probe and the fourthprobe contains both (i) a fluorescent dye and (ii) a quencher dye whichis capable of quenching fluorescence emission from the fluorescent dyewhen the fluorescent dye is subjected to fluorescence excitation energy,and said cleaving severs a covalent linkage between the fluorescent dyeand the quencher dye in the second probe and/or fourth probe, therebyincreasing an observable fluorescence signal from the fluorescent dye.In some embodiments, the second probe and the fourth probe can eachcontain (i) a fluorescent dye and (ii) a quencher dye.

In some embodiments, the step of cleaving further produces a secondfragment from the fourth probe that does not associate with the fourthhybridization complex, and the method further includes detecting bothsecond fragments. In some embodiments, the second fragment comprises oneor more contiguous nucleotides that are substantially non-complementaryto the target strand. In some embodiments, one or more contiguousnucleotides comprise 1 to 20 nucleotides.

In some embodiments, the method further includes immobilizing the secondfragment on a solid support. In some embodiments, the method furtherincludes subjecting the second fragment to electrophoresis. In someembodiments, the method further includes detecting the second fragmentby mass spectrometry. In some embodiments, the method further comprisesdetecting the second fragment after the last cycle. In some embodiments,the method further comprises detecting the second fragment during orafter a plurality of cycles. In some embodiments, the method furthercomprises detecting the second fragment during all of the cycles. Insome embodiments, the method further includes detecting the firsthybridization complex, the second hybridization complex, or both, afterat least one cycle. In some embodiments, the method further includesdetecting the third hybridization complex, the fourth hybridizationcomplex, or both, after at least one cycle. In some embodiments, themethod further includes detecting the first ligated strand, the secondligated strand, or both, after at least one cycle. In some embodiments,said detecting comprises an electrophoretic separation step.

Further embodiments of the ligation method for detecting a targetpolynucleotide can be found in Bi, W., et al., U.S. Pat. No. 6,511,810incorporated herein by reference in its entirety.

EXAMPLES Materials and Methods

Unless otherwise indicated, all synthesis reactions were carried out inoven or flame dried glassware, under an atmosphere of Argon.Tetrahydrofuran (THF) and methylene chloride (CH₂Cl₂) were distilledfrom calcium hydride (CaH₂) under Argon. Unless otherwise indicated, allother solvents were used as received from the distributor. Thin layerchromatography (TLC) was performed on 1 mm silica gel plates purchasedfrom Sigma-Aldrich (Milwaukee, Wis.) and visualized with UV light(Spectroline; model ENF-240C) or stained with KMnO₄ or phosphomolybdicacid. Flash column chromatography was performed using silica gel with anaverage particle size of 40 μm purchased from Sigma-Aldrich (Milwaukee,Wis.). 5-Cyano-2′-deoxyuridine was synthesized according to Hampton, etal., J. Med. Chem., v.22 (6), 621-631 (1979). 5-Fluoro-2′-deoxyuridineCPG support was purchased from ChemGenes Corp. (Wilmington, Mass.).Non-derivitized CPG support was obtained from Applied Biosystems Inc(P/N 360139, Foster City, Calif.). Unless otherwise indicated, all otherreagents were purchased from Sigma-Aldrich. Unless otherwise indicated,automated DNA synthesis was carried out on an ABI 394 DNA synthesizer ata 0.2 mmol scale following the standard protocol. Oligonucleotides werepurified by reversed phase HPLC on an Agilent 1100 HPLC system. ESI-TOFmass spectra were recorded on a Mariner mass spectrometer (AppliedBiosystems, Foster City). The purity of synthesized oligo-nucleotideswas checked by capillary electrophoresis (CE) on an Agilent CE system.

Oligonucleotide Synthesis 5-Cyano-2′-deoxyuridine CPG and5-trifluoromethyl-2′-deoxyuridine CPG

5-Cyano-2′-deoxyuridine CPG and 5-trifluoromethyl-2′-deoxyuridine CPGwere synthesized according Scheme 1.

5-Trifluoromethyl-5′-dimethoxytrityl-2′-deoxyuridine

To a stirred solution of 250 mg of 5-trifluoromethyl-2′ deoxyuridine(5-Tf-dU) in 20 mL of dry pyridine at 0° C. was added 227 mg ofdimethoxytrityl chloride (DMT-Cl) and 10 mg of 4-dimethylaminopyridine(DMAP). The reaction was stirred for 5 hours at room temperature when asecond portion of 144 mg of DMT-Cl was added. After stirring for 12hours at room temperature, a third portion of 100 mg of DMT-Cl was addedto the reaction. The reaction was stirred for an additional 4 hours atroom temperature and then diluted with CH₂Cl₂, washed with water anddried over sodium sulfate (Na₂SO₄). The crude product was purified bysilica gel column chromatography (0-10% methanol (MeOH) in CH₂Cl₂). 485mg of the desired product (5′-DMT-5-Tf-dU) was obtained as a yellow foam(96% yield).

5-Trifluoromethyl-4′-(3-carboxypropionyl)-5′-dimethoxytrityl-2′-deoxyuridine

To a stirred solution of 470 mg of DMT-5-TfU, obtained above, in 20 mLof CH₂Cl₂ was added 133 mg of succinic anhydride, 48 mg of DMAP and 217μL of triethylamine (Et₃N). After stirring for 12 hrs., the mixture waswashed with 5% aqueous citric acid solution (1×30 mL) and saturatedsodium chloride, NaCl, (1×30 mL). The organic layer was dried overNa₂SO₄ and evaporated to give 500 mg of5-trifluoromethyl-4′-(3-carboxypropionyl)-5′-dimethoxytrityl-2′-deoxyuridineas a pale yellow foam.

5-Trifluoromethyl-2′-deoxyuridine CPG

To a solution of 400 mg of5-trifluoromethyl-4′-(3-carboxypropionyl)-5′-dimethoxytrityl-2′-deoxyuridinein 20 mL of dimethylformamide (DMF) was added 98 mg of2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), 135 μL of diisopropylethyl amine (DIPEA) and 7 g of CPG (37μM/g). The mixture was shaken for 16 hours and then washed with DMF(3×50 mL), THF (3×30 mL) acetonitrile, CH₃CN, (2×30 mL) and CH₂Cl₂ (2×30mL). The 5-trifluoromethyl-2′-deoxyuridine CPG product was dried underhigh vacuum for 4 hours.

5-Cyano-5′-dimethoxytrityl-2′-deoxyuridine

To a stirred solution of 506 mg of 5-cyano-2′ deoxyuridine (5-CN-dU) in124 mL of dry pyridine at 0° C. was added 813 mg of DMT-Cl and 49 mg ofDMAP. The reaction was stirred for 12 hours at room temperature and thendiluted with CH₂Cl₂, washed with water and dried over sodium sulfate(Na₂SO₄). The crude product was purified by silica gel columnchromatography (10% MeOH in CH₂Cl₂). 710 mg of the desired product(5′-DMT-5-CN-dU) was obtained (64% yield).

5-Cyano-4′-(3-carboxypropionyl)-5′-dimethoxytrityl-2′-deoxyuridine

To a stirred solution of 310 mg of DMT-5-CN-dU, obtained above, in 7.5mL of CH₂Cl₂ was added 100 mg of succinic anhydride, 34 mg of DMAP and167 μL of Et₃N. After stirring for 12 hours at room temperature, thesolvent was evaporated to give crude5-Cyano-4′-(3-carboxypropionyl)-5′-dimethoxytrityl-2′-deoxyuridine whichwas used in the next step without further purification.

5-Cyano-2′-deoxyuridine CPG

To a solution of crude5-trifluoromethyl-4′-(3-carboxypropionyl)-5′-dimethoxytrityl-2′-deoxyuridinein 15 mL of DMF was added 212 mg of HBTU, 145 μL of DIPEA and 7.5 g ofCPG (37 μM/g). The mixture was stirred for 1 hour and then washed withDMF (3×50 mL), THF (3×30 mL) and CH₂Cl₂ (2×30 mL). The5-trifluoromethyl-2′-deoxyuridine CPG product was dried under highvacuum.

3′-Terminal oligonucleotides of 5-cyano-2′-deoxyuridine,5-trifluoromethyl-2′-deoxyuridine, and 5-fluoro-2′-deoxyuridine

As mentioned above, automated DNA synthesis was carried out on an ABI394 DNA synthesizer at a 0.2 μmol scale following the standard protocol.Oligonucleotides were purified by reversed phase HPLC on an Agilent 1100HPLC system. To that end, the oligonucleotides shown in Tables 1-4 weresynthesized. Forward primer AGTFcT and reverse primer AGTRcT amplifypart of the human angiotesinogen gene (AGT, 125 bp). Forward primerLIGFcT and reverse primer LIGRcT amplify part of the human DNA ligase Igene (LIG, 82 bp). Forward primer D10SFcT and reverse primer D10SRcTamplify part of human chromosome 10 clone RP11-143D9 (D10S, 102 bp).

TABLE 1  Primers X = 5-Cyano-2′- X = T deoxyuridine Primer SequencesAGTFcT AGTFcT-CN GGTCAGTTAATAACCACCTTTCACCCX SEQ ID NO: 1 AGTRcTAGTRcT-CN GCCAGGAGGCAGAGGATGGX SEQ ID NO: 2 LIGFcT LIGFcT-CNGGAGACCCCGAAAGAAAGCCX SEQ ID NO: 3 LIGRcT LIGRcT-CN AGGCGTGGTGGGCTGGXSEQ ID NO: 4 D10SFcT D10SFcT-CN CATATCTCACTCCTAAAACCCACAGGX SEQ ID NO: 5D10SRcT D10SRcT-CN CAGACACCTACCACCTGCCCX SEQ ID NO: 6

TABLE 2  Primers X = 5-Trifluoromethyl- X = T 2′-deoxyuridinePrimer Sequences LIGFcT LIGFcT-CF₃ GGAGACCCCGAAAGAAAGCCX SEQ ID NO: 7LIGRcT LIGRcT-CF₃ AGGCGTGGTGGGCTGGX SEQ ID NO: 8

TABLE 3  Primers X = 5-Fluoro-2′- X = T deoxyuridine Primer SequencesAGTFcT AGTFcT-F GGTCAGTTAATAACCACCTTTCACCCX SEQ ID NO: 9 AGTRcT AGTRcT-FGCCAGGAGGCAGAGGATGGX SEQ ID NO: 10 LIGFcT LIGFcT-F GGAGACCCCGAAAGAAAGCCXSEQ ID NO: 11 LIGRcT LIGRcT-F AGGCGTGGTGGGCTGGX SEQ ID NO: 12

TABLE 4  Primers X = 5-CN-2′- X = T deoxyuridine Primer SequencesLamda-FcG Lamda-FcG-5-CN ATCAGAAACGAACGCATCATCAAGX SEQ ID NO: 13Lamda-RcG Lamda-RcG-5-CN AAACAGCCACAAAGCCAGCCGGAAX SEQ ID NO: 14

A second set of primers designed for amplification of AGT were preparedas described above. Each set having a 3′ terminus of either 5-CN-dU,5-CF₃-dU or 5-F-dU as shown below in Tables 4-6.

TABLE 5  Primers X = 5-Cyano-2′- X = T deoxyuridine Primer SequencesAGTFcT AGTFcT-CN GCTCTCTGGACTTCACAGAACTGGAX SEQ ID NO: 15 AGTRcTAGTRcT-CN CCTTACCTTGGAAGTGGACGTAGGX SEQ ID NO: 16

TABLE 6  Primers X = 5-CF₃-2′- X = T deoxyuridine Primer SequencesAGTFcT AGTFcT-CF₃ GCTCTCTGGACTTCACAGAACTGGAX SEQ ID NO: 17 AGTRcTAGTRcT-CF₃ CCTTACCTTGGAAGTGGACGTAGGX SEQ ID NO: 18

TABLE 7  Primers X = 5-Fluoro-2′- X = T deoxyuridine Primer SequencesAGTFcT AGTFcT-F GCTCTCTGGACTTCACAGAACTGGAX SEQ ID NO: 19 AGTRcT AGTRcT-FCCTTACCTTGGAAGTGGACGTAGGX SEQ ID NO: 20

5-Cyano-2′-deoxyuridine phosphoramidite

To a stirred solution of 250 mg of 5′-DMT-5-CN-dU in 20 mL CH₂Cl₂ wasadded 8 mg of tetrazole-triethylamine and 190 μL of2-cyanoethyl-tetraisopropylphosphoramidite ([(i-Pr)₂N]₂POCH₂CH₂CN). Themixture was stirred at room temperature for 12 hours and the solventremoved under vacuum. The crude product was purified by silica gelchromatography (25/25/1 CH₂Cl₂/CH₃CN/Et₃N) to give 290 mg of5-cyano-2′-deoxyuridine phosphoramidite (85% yield).

5-cyano-2′-deoxyuridine Containing Oligonucleotides

As mentioned above, automated DNA synthesis was carried out on an ABI394 DNA synthesizer at a 0.2 μmol scale following the standard protocol.Oligonucleotides were purified by reversed phase HPLC on an Agilent 1100HPLC system. To that end, the oligonucleotides shown in rows 2, 3 and 5of Tables 7-8 were synthesized using 5-cyano-2′-deoxyuridinephosphoramidite to incorporate 5-cyano-2′-deoxyuridine at positionsother than the 3′ terminus, and the oligonucleotides having5-cyano-2′-deoxyuridine at the 3′ terminus were synthesized as describedabove.

TABLE 8  Primers X = 5-Cyano-2′- X = T deoxyuridine Primer SequencesAGTFcT-short AGTFcT-short-1-CN GGTCAGTTAATAACCACCTTX SEQ ID NO: 21AGTFcT-short-2-CN GGTCAGTTAATAACCACCTXT SEQ ID NO: 22 AGTFcT-short-3-CNGGTCAGTTAATAACCACCXTT SEQ ID NO: 23 AGTRcT AGTRcT-CNGCCAGGAGGCAGAGGATGGX SEQ ID NO: 24 AGTRcT-4-CN GCCAGGAGGCAGAGGAXGGTSEQ ID NO: 25

TABLE 9  Primers X = 5-Cyano-2′- X = T deoxyuridine Primer SequencesAGTFcT AGTFcT-1-CN CTCACCCTCATGGCCTCATTX SEQ ID NO: 26 AGTFcT-2-CNCTCACCCTCATGGCCTCATXT SEQ ID NO: 27 AGTFcT-3-CN CTCACCCTCATGGCCTCAXTTSEQ ID NO: 28 AGTRcT-4-CN ACCTCCCCAACGGCCAXAAT SEQ ID NO: 29

Amplifications:

All gDNA amplifications were carried out with 5000 copies of gDNA per 15μL, reaction and all X-DNA amplifications were carried out with 1500copies of gDNA per 15 μL, reaction.

Amplification Reaction Condition #1:

Amplifications were carried out in 15 μL, reactions using eitherunmodified and/or modified primers containing the following reagents:

SYBR® Green PCR Master Mix (Applied Biosystems, P/N 4309155)

900 nmoles of each forward and reverse primer

water for no template control (NTC) experiments or 15 ng genomic DNA(Applied Biosystems, P/N 403062)

Amplification Reaction Condition #2:

Amplifications were carried out in 15 μL reactions using eitherunmodified and/or modified primers containing the following reagents:

10 mM Tris-HCl buffer, pH 8.3

50 mM KCl, 3 mM MgCl₂, 0.01% v/v gelatin

0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, and 0.4 mM dUTP

0.025 unit/4 AmpliTaq Gold® DNA polymerase (Applied Biosystems, P/NN808-0107)

200 nM of each forward and reverse primer

water for no template control (NTC) experiments or 15 ng genomic DNA(Applied Biosystems, P/N 403062)

Amplification Reaction Condition #3:

Amplifications were carried out in 15 μL reactions using eitherunmodified and/or modified primers containing the following reagents:

10 mM Tris-HCl buffer, pH 8.3

50 mM KCl, 3 mM MgCl₂, 0.01% v/v gelatin

0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, and 0.4 mM dUTP

0.025 unit/μL AmpliTaq® DNA polymerase (Applied Biosystems, P/NN808-0158)

200 nM of each forward and reverse primer

water for no template control (NTC) experiments or 15 ng genomic DNA(Applied Biosystems, P/N 403062)

The progress of all PCR reactions was monitored in real time by SYBR®Green assay on an ABI PRISM 7900HT Sequence Detection System (AppliedBiosystems; Foster City, Calif.) as described in SYBR® Green PCR MasterMix and RT-PCR: Protocol (Applied Biosystems, 2002).

Amplification Reaction Analysis:

All amplification products were analyzed by gel electrophoresis asfollows. PCR reaction mixture (15 μL) was diluted with 5 μLnuclease-free water. This solution was loaded along with 25 by DNAladder into wells of 4% agarose gel (Invitrogen, P/N G5018-04).Electrophoresis was carried out at 12 V DC at 880 mA for 30 minutes.Ethidium bromide stained bands of dsDNA were visualized using UVirradiation and quantified by AlphaDigDoc 1000 software (Alpha Innotech;San Leandro, Calif.) equipped with a Kodak digital camera.

Thermal Cycling:

Thermal Cycling Protocol #1:

Amplification temperature cycling was carried out using the followingthermal cycling protocol.

TABLE 10 AmpliTaq Pre-reaction Gold ® 50 Cycles incubation ActivationDenature Anneal/Extend Temperature 50 95 95 60 72 (° C.) Time 2 min 10min 15 sec 30 sec 30 sec

Thermal Cycling Protocol #2:

Amplification temperature cycling was carried out using the followingthermal cycling protocol.

TABLE 11 AmpliTaq Gold ® 50 Cycles Activation Denature Anneal/ExtendTemperature (° C.) 95 95 60 72 Time 10 min 15 sec 30 sec 30 sec

Thermal Cycling Protocol #3:

Amplification temperature cycling was carried out using the followingthermal cycling protocol.

TABLE 12 AmpliTaq Gold ® 50 Cycles Activation Denature Anneal/ExtendTemperature (° C.) 95 95 60 72 Time 2 min 15 sec 30 sec 30 sec

Thermal Cycling Protocol #4:

Amplification temperature cycling was carried out using the followingthermal cycling protocol.

TABLE 13 AmpliTaq Gold ® 50 Cycles Activation Denature Anneal/ExtendTemperature (° C.) 93 93 60 72 Time 2 min 60 sec 50 sec 90 sec

Amplifications using 3′-Terminus Modified Primers:

5-Cyano-2′-deoxyuridine 3′-Terminal Primers/AmpliTaq Gold®:

Amplification reactions using 3′-terminal 5-cyano-2′-deoxyuridinecontaining primers, shown in Table 1, were carried out underAmplification Reaction Condition #1 according to Thermal CyclingProtocol #1.

For each primer pair, amplification reactions using unmodified and3′-modified primers were run with gDNA corresponding to the primer pair.Additionally, amplification reactions using unmodified and 3′-modifiedprimers were run without any template as no template control (NTC)reactions.

Gel electrophoresis analysis was carried out as described above. Gelelectrophoresis showed a significant amount of non-specificamplification product at about 40-50 bp, depending on the primer setused, in NTC amplifications with unmodified primers indicating theformation of primer-dimer amplicons. On the otherhand, significantlydecreased primer-dimer amplicon formation was observed in gelelectrophoresis images of NTC amplifications using 3′-modified primers.Gel electrophoresis of gDNA template amplification reactions usingunmodified primers clearly showed a band corresponding to the desiredtemplate amplicon and also showed a band at corresponding toprimer-dimer amplicon formation.

Amplification of AGT template using unmodified primers clearly showed aband at about 125 by corresponding to the desired template amplicon andalso showed a band at about 50 by corresponding to primer-dimer ampliconformation. On the otherhand, amplification of AGT template using3′-modified primers clearly showed a band at about 125 by correspondingto the desired template amplicon but also showed significantly decreasedprimer-dimer amplicon formation.

Real time PCR monitoring showed that the AGT gDNA amplificationefficiencies using unmodified- and 3′-modified-primers were similar. Onthe otherhand, while the NTC amplification using unmodified primers gavea C_(t) value of about 37, NTC amplification using 5-cyano-2′-dUmodified primers did not give a measurable signal until after the44^(th) cycle.

Amplification of LIG template using unmodified primers clearly showed aband at about 80 by corresponding to the desired template amplicon andalso showed a band at about 40 by corresponding to primer-dimer ampliconformation. On the otherhand, amplification of LIG template using3′-modified primers clearly showed a band at about 80 by correspondingto the desired template amplicon but also showed significantly decreasedprimer-dimer amplicon formation.

Amplification of D10S template using unmodified primers clearly showed aband at about 100 by corresponding to the desired template amplicon andalso showed a band at about 50 by corresponding to primer-dimer ampliconformation. On the otherhand, amplification of D10S template using3′-modified primers clearly showed a band at about 100 by correspondingto the desired template amplicon but also showed significantly decreasedprimer-dimer amplicon formation.

Real time PCR monitoring showed similar results. In all cases, C_(t)values for NTC amplifications with 3′-modified primers weresignificantly higher than C_(t) values for NTC amplifications usingunmodified primers indicating a suppression of primer-dimeramplification. In addition, C_(t) values for each desired amplicon weresimilar between the unmodified and 3′ modified primers indicating thatamplification efficiency was not compromised by 3′ modification.

5-Trifluoromethyl-2′-deoxyuridine 3′-Terminal Primers/AmpliTaq Gold®:

Amplification reactions using 3′-terminal5-trifluoromethyl-2′-deoxyuridine containing primers, shown in Table 2,were carried out under Amplification Reaction Condition #1 according toThermal Cycling Protocol #1. Forward primer LIGFcT and reverse primerLIGRcT amplify part of the human DNA ligase I gene (LIG, 82 bp).

Amplification reactions using unmodified and 3′-modified primers wererun with gDNA. Additionally, amplification reactions using unmodifiedand 3′-modified primers were run without any template as no templatecontrol (NTC) reactions.

Gel electrophoresis analysis was carried out as described above. Gelelectrophoresis showed a significant amount of non-specificamplification product at about 40 by in NTC amplifications withunmodified primers indicating the formation of primer-dimer amplicons.On the otherhand, significantly decreased primer-dimer ampliconformation was observed in gel electrophoresis images of NTCamplifications using 3′-modified primers. Gel electrophoresis of gDNAtemplate amplification reactions using unmodified primers clearly showeda band corresponding to the desired template amplicon and also showed aband at corresponding to primer-dimer amplicon formation.

Amplification of LIG template using unmodified primers clearly showed aband at about 80 by corresponding to the desired template amplicon andalso showed a band at about 40 by corresponding to primer-dimer ampliconformation. On the otherhand, amplification of LIG template using3′-modified primers clearly showed a band at about 80 by correspondingto the desired template amplicon but also showed significantly decreasedprimer-dimer amplicon formation.

Real time PCR monitoring showed that the LIG gDNA amplificationefficiencies using unmodified- and 3′-modified-primers were similar. Onthe otherhand, while the NTC amplification using unmodified primers gavea C_(t) value of about 40, NTC amplification using5-trifluoromethyl-2′-dU modified primers provided no measurable signalthrough the 50-cycle amplification.

5-Fluoro-2′-deoxyuridine 3′-Terminal Primers/AmpliTaq Gold®:

Amplification reactions using 3′-terminal 5-fluoro-2′-deoxyuridinecontaining primers, shown in Table 3, were carried out underAmplification Reaction Condition #1 according to Thermal CyclingProtocol #2. Forward primer AGTFcT and reverse primer AGTRcT amplifypart of the human angiotesinogen gene (AGT, 125 bp). Forward primerLIGFcT and reverse primer LIGRcT amplify part of the human DNA ligase Igene (LIG, 82 bp).

For each primer pair, amplification reactions using unmodified and3′-modified primers were run with gDNA corresponding to the primer pair.Additionally, amplification reactions using unmodified and 3′-modifiedprimers were run without any template as no template control (NTC)reactions.

Gel electrophoresis analysis was carried out as described above. Gelelectrophoresis showed a significant amount of non-specificamplification product at about 40-50 bp, depending on the primer setused, in NTC amplifications with unmodified primers indicating theformation of primer-dimer amplicons. On the otherhand, significantlydecreased primer-dimer amplicon formation was observed in gelelectrophoresis images of NTC amplifications using 3′-modified primers.Gel electrophoresis of gDNA template amplification reactions usingunmodified primers clearly showed a band corresponding to the desiredtemplate amplicon and also showed a band at corresponding toprimer-dimer amplicon formation.

Amplification of AGT template using unmodified primers clearly showed aband at about 125 by corresponding to the desired template amplicon andalso showed a band at about 50 by corresponding to primer-dimer ampliconformation. On the otherhand, amplification of AGT template using3′-modified primers clearly showed a band at about 125 by correspondingto the desired template amplicon but also showed significantly decreasedprimer-dimer amplicon formation.

Real time PCR monitoring showed that the AGT gDNA amplificationefficiencies using unmodified- and 3′-modified-primers were verysimilar. On the otherhand, while the NTC amplification using unmodifiedprimers gave a C_(t) value of about 36, NTC amplification using5-fluoro-2′-dU modified primers did not give a measurable signal untilalmost the 40^(th) cycle.

Amplification of LIG template using unmodified primers clearly showed aband at about 80 by corresponding to the desired template amplicon andalso showed a band at about 40 by corresponding to primer-dimer ampliconformation. On the otherhand, amplification of LIG template using3′-modified primers clearly showed a band at about 80 by correspondingto the desired template amplicon but also showed significantly decreasedprimer-dimer amplicon formation.

Real time PCR monitoring showed that the LIG gDNA amplificationefficiencies using unmodified- and 3′-modified-primers were similar. Onthe otherhand, while the NTC amplification using unmodified primers gavea C_(t) value of about 40, NTC amplification using 5-fluoro-2′-dUmodified primers did not give a measurable signal until after the49^(th) cycle.

5-Cyano-2′-deoxyuridine Containing Primers/AmpliTaq Gold®:

Amplification reactions using 5-cyano-2′-deoxyuridine containingprimers, shown in Table 8, were carried out under Amplification ReactionCondition #1 according to Thermal Cycling Protocol #2.

For each primer pair, amplification reactions using unmodified andmodified primers were run with gDNA corresponding to the primer pair.Additionally, amplification reactions using unmodified and 3′-modifiedprimers were run without any template as no template control (NTC)reactions.

Gel electrophoresis analysis was carried out as described above. Gelelectrophoresis of amplification using unmodified AGTFcT-short primershowed a significant amount of non-specific amplification product atabout 50 by in NTC amplifications indicating the formation ofprimer-dimer amplicons. On the otherhand, no primer-dimer ampliconformation was observed in gel electrophoresis images of NTCamplifications using AGTFcT-short-1-CN, AGTFcT-short-2-CN,AGTFcT-short-3-CN modified primers.

Real time PCR monitoring of NTC amplification using unmodified primersgave a C_(t) value of about 34. On the other hand, NTC amplificationusing 5-cyano-2′-dU modified primers AGTFcT-short-1-CN,AGTFcT-short-2-CN, and AGTFcT-short-3-CN gave no measurable signalthrough the 50-cycle amplification.

Amplification of AGT template using AGTFcT-short-1-CN,AGTFcT-short-2-CN, AGTFcT-short-3-CN modified primers clearly showed aband at about 125 by corresponding to the desired template amplicon butalso showed no primer-dimer amplicon formation.

Real time PCR monitoring showed that the AGT gDNA amplificationefficiencies using unmodified- and 5-CN-dU containing primersAGTFcT-short-1-CN, AGTFcT-short-2-CN and AGTFcT-short-3-CN were similar,with all C_(t) values falling between 26 and 29 cycles.

Gel electrophoresis of amplification using unmodified AGT primer showeda significant amount of non-specific amplification products betweenabout 40 by and 80 by in NTC amplifications indicating the formation ofprimer-dimer amplicons. On the otherhand, primer-dimer ampliconformation was significantly decreased in gel electrophoresis images ofNTC amplifications using AGTFcT-4-CN modified primer.

Likewise in the AGT gDNA template amplification, gel electrophoresis ofamplification using unmodified AGTFcT primer showed a band at about 125by attributed to the desired amplicon as well as a significant amount ofnon-specific amplification products between about 50 by and 80 by ingDNA amplifications indicating the formation of primer-dimer amplicons.On the otherhand, primer-dimer amplicon formation was not seen in gelelectrophoresis images of NTC amplifications using AGTFcT-4-CN modifiedprimers.

Real time PCR monitoring showed that the AGT gDNA amplificationefficiencies using unmodified- and 5-CN-dU containing primer AGTFcT-4-CNwere very similar, with C_(t) values 28 and 29 cycles respectively. Realtime monitoring of the NTC amplifications of unmodified-AGT andAGTFcT-4-CN primers gave C_(t) values of 38 and 43 cycles respectively.

5-Cyano-2′-deoxyuridine, 5-CF₃-2-deoxyuridine and5-Fluoro-2′deoxyuridine 3′-Terminal Modified Primers/AmpliTaq®:

Amplification reactions using 3′-terminal modified primers, shown inTable 5-7, were carried out under Amplification Reaction Condition #3according to Thermal Cycling Protocol #3.

For each primer pair, amplification reactions using unmodified and3′-modified primers were run with gDNA corresponding to the primer pair.Additionally, amplification reactions using unmodified and 3′-modifiedprimers were run without any template as no template control (NTC)reactions.

Gel electrophoresis analysis was carried out as described above. Gelelectrophoresis of amplification using unmodified primer showed asignificant amount of non-specific amplification product at about 60 byin NTC amplifications with unmodified primers indicating the formationof primer-dimer amplicons. On the otherhand, no primer-dimer ampliconformation was observed in gel electrophoresis images of NTCamplifications using 3′-terminal modified primers.

Amplification of AGT template using 5-CN-dU, 5-CF₃-dU and 5-F-dU3′-terminal modified primers clearly showed a band at about 125 bycorresponding to the desired template amplicon but also showed noprimer-dimer amplicon formation.

5-Cyano-2′-deoxyuridine Containing Primers/AmpliTaq®:

Amplification reactions using 5-cyano-2′-deoxyuridine containingprimers, shown in Table 9, were carried out under Amplification ReactionCondition #3 according to Thermal Cycling Protocol #3.

For each primer pair, amplification reactions using unmodified andmodified primers were run with gDNA corresponding to the primer pair.Additionally, amplification reactions using unmodified and 3′-modifiedprimers were run without any template as no template control (NTC)reactions.

Gel electrophoresis analysis was carried out as described above. Gelelectrophoresis of amplification using unmodified AGTFcT primer showed asignificant amount of non-specific amplification product at about 50 byin NTC amplifications with unmodified primers indicating the formationof primer-dimer amplicons. On the otherhand, no primer-dimer ampliconformation was observed in gel electrophoresis images of NTCamplifications using AGTFcT-1-CN, AGTFcT-2-CN, AGTFcT-3-CN orAGTRcT-4-CN modified primers.

Amplification of AGT template using AGTFcT-1-CN, AGTFcT-2-CN,AGTFcT-3-CN or AGTRcT-4-CN modified primers clearly showed a band atabout 125 by corresponding to the desired template amplicon but alsoshowed no primer-dimer amplicon formation.

λ-DNA Amplifications:

To test whether 3′-modified primers of the present teachings couldreduce non-specific amplification without effecting amplificationefficiency of longer amplicons, X-DNA was used as a template withprimers designed to make a 1700 by target amplicon.

5-Cyano-2′-deoxyuridine λ-DNA Amplification:

Amplification reactions using 3′-terminal 5-cyano-2′-deoxyuridinecontaining primers, shown in Table 4, were carried out underAmplification Reaction Condition #2 according to Thermal CyclingProtocol #4. Both real time analysis and gel electrophoresis showed thateven with longer amplicons, 3′-modification with 5-CN-2′-deoxyuridineresulted in significant reduction in primer-dimer amplification withoutadversely effecting target amplification efficiency.

1.-110. (canceled)
 111. A polynucleotide comprising: at least onemodified pyrimidine nucleobase of the structure:

wherein X is N or C; wherein at least one of R¹ or R² is an electronwithdrawing substituent or X is N, such that when X is N, R² is absent;and at least one said modified pyrimidine nucleobase is no more than 4nucleotides from the 3′ terminus of the polynucleotide, thepolynucleotide operable to reduce primer-dimer formation in nucleic acidamplification reactions.
 112. The polynucleotide according to claim 111,wherein R¹ is —CN.
 113. The polynucleotide according to claim 111,wherein R¹ is —F.
 114. The polynucleotide according to claim 111,wherein R¹ is —CF₃.
 115. The polynucleotide according to claim 111,wherein R² is —CN.
 116. The polynucleotide according to claim 111,wherein R² is —F.
 117. The polynucleotide according to claim 111,wherein R² is —CF₃.
 118. The polynucleotide according to claim 111,wherein X is C.
 119. The polynucleotide according to claim 118, whereinR² is —H.
 120. The polynucleotide according to claim 119, wherein R¹ is—CN.
 121. The polynucleotide according to claim 119, wherein R¹ is —F.122. The polynucleotide according to claim 119, wherein R¹ is —CF₃. 123.The polynucleotide according to claim 118, wherein R² is —CN.
 124. Thepolynucleotide according to claim 118, wherein R² is —F.
 125. Thepolynucleotide according to claim 118, wherein R² is —CF₃.
 126. Thepolynucleotide according to claim 111, wherein X is N.
 127. Thepolynucleotide according to claim 126, wherein R¹ is —CN.
 128. Thepolynucleotide according to claim 126, wherein R¹ is —F.
 129. Thepolynucleotide according to claim 126, wherein R¹ is —CF₃.
 130. A methodfor reducing formation of primer-dimers comprising: amplifying a targetnucleic acid using at least two primers having at least one modifiedpyrimidine nucleobase having a structure comprising:

wherein X is N or C; wherein at least one of R¹ or R² is an electronwithdrawing substituent or X is N, such that when X is N, R² is absent;and at least one said modified pyrimidine nucleobase is no more than 4nucleotides from the 3′ terminus of the polynucleotide, thepolynucleotide operable to reduce primer-dimer formation in nucleic acidamplification reactions.
 131. The method of claim 130, wherein the atleast one modified pyrimidine nucleobase has a structure comprising:


132. The method of claim 130, wherein the at least one modifiedpyrimidine nucleobase has a structure comprising:


133. The method of claim 130, wherein the at least one modifiedpyrimidine nucleobase has a structure comprising: